Default apoptotic programs have been adapted as a first line of defense against cancer, and virtually all cancer cells have mutations that activate survival signals in order to suppress apoptosis. A central node in survival signaling is mTOR (the mammalian target of rapamycin). mTOR is activated in response to signals mediated by the phosphatidylinositol-3-kinase (PI3K) signaling pathway. However, more recently it has become apparent that mTOR is also targeted by signals that activate phospholipase D (PLD). PLD generates phosphatidic acid (PA), a lipid second messenger that interacts directly with mTOR in a manner that is competitive with rapamycin - and PA is required for the activation of mTOR. Importantly, PLD activity is elevated in several types of human cancer. PLD activity is elevated in many human cancer cells and is required for mTOR-mediated signals that are critical for survival and promote cell cycle progression. The CENTRAL HYPOTHESIS of the proposal is - Elevated PLD activity in human cancer cells promotes passage through a late G1 Cell Growth Checkpoint and suppresses default apoptotic programs. We are proposing that virtually all cancer cells must activate signals that allow passage through this checkpoint. SPECIFICALLY, we propose: 1) To determine how PLD-mTOR signaling impacts on cell cycle progression through a proposed Cell Growth Checkpoint; 2) To determine the mechanism by which PLD-generated PA regulates mTORC1 and mTORC2 in concert with other signaling inputs; and 3) To characterize signals that lead to elevated PLD activity in human cancer cell lines and to evaluate targeting these signals pharmacologically both in vitro and in vivo. We are proposing that a PLD-mTOR signaling pathway in human cancer cells represents a widely employed strategy by cancer cells to promote cell cycle progression and suppress default apoptotic programs. The studies proposed here will provide a framework for the rational targeting of an apparent large number of cancers that depend upon elevated PLD activity for G1 cell cycle progression and suppression of apoptosis. PUBLIC HEALTH RELEVANCE: The proposed study will evaluate the intracellular mechanisms that regulate phospholipase D (PLD) and mTOR in human cancer cells and the impact of these signals on a proposed Cell Growth Checkpoint in the G1 phase of the cell cycle that we are proposing must be overcome in virtually all human cancers. The project builds on our previous findings that there is elevated phospholipase D (PLD) activity in many human cancer cells and that suppression of PLD activity in these cells results in apoptotic cell death. Targeting the PLD-mTOR signals in cancer cells represents a very promising strategy for resurrecting the default cell death programs that are arguably the first line of defense against cancer - and is therefore a fertile area for translational research.